Capsaicin combined with dietary fiber prevents high‐fat diet associated aberrant lipid metabolism by improving the structure of intestinal flora

Abstract Capsaicin (CAP) and dietary fibers are natural active ingredients that given separately do positively affect obesity and metabolic diseases. However, it was unknown whether their combined administration might further improve blood lipids and gut flora composition. To test this hypothesis we administered capsaicin plus dietary fibers (CAP + DFs) to male rats on a high‐fat diet and analyzed any changes in the intestinal microbiota make up, metabolites, and blood indexes. Our results showed that combining CAP with dietary fibers more intensely reduced total cholesterol (TC) and low‐density lipoprotein cholesterol (LDL‐C). CAP + DFs also increased gut bacteria variety, and the abundance of several beneficial bacterial strains, including Allobaculum and Akkermansia, while reducing harmful strains such as Desulfovibrio. Additionally, CAP + DFs significantly increased arginine levels and caused short‐chain fatty acids accumulation in the contents of the cecal portion of rats' gut. In conclusion, notwithstanding the rats were kept on a high‐fat diet, adding CAP + DFs to the chow further improved, as compared with CAP alone, the lipidemia and increased the gut beneficial bacterial strains, while reducing the harmful ones.


| Animals and treatments
(200 ± 20 g) (from Hunan SJA Laboratory Animal Co., Ltd) were kept inside individual stainless steel cages at 25 ± 1°C, with a 40%-70% a relative humidity and a 12 h light/dark cycle. In the week preceding the experiments, rats had an ad libitum access to water and were kept on a standard diet. A total of 30 rats were randomly divided into three groups (n = 10): (i) high-fat diet (HF); (ii) CAP 0.01% + high-fat diet (HFC); (iii) CAP0.01% + 5%DFs + high-fat diet (HFBM). Table S1 lists each experimental group's diet ingredients. The high-cholesterol food contained a 1% cholesterol supplement based on U.S. Standard AIN-93 feed formula (Afrose et al., 2009). Food treated with Co 60 irradiation for sterilization was purchased from the Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd. The sterilized formulations were stored in a refrigerator (−20°C) until use.

| Sample collection
The rats' survival and food intake were checked every day. The rats' weight was recorded once weekly. After 4 weeks of feeding, rats were fasted for one day and next sacrificed. Immediately after sacrifice, their blood, cecum with its contents, and small intestine with its contents were collected under aseptic conditions. All samples were immediately placed in sterile tubes, flash-frozen (30 s) in liquid N, and next stored at −80°C.

| Blood biochemical analyses
Plasma triglycerides (TG) and cholesterol (TC) levels were deter-
The retention time of each chromatographic peak was used for qualitative analysis, and the resulting peak areas for different standard concentrations served for the quantitations. The GC chromatogram of SCFA standards is shown in Figure S1, while the standard curves are provided in Table S2. 2.6 | Gut microbiota analysis by 16S rRNA gene sequencing DNA preparation, PCR amplification, and pyrosequencing were performed according to Kørner et al. (2015). Briefly, total DNA was extracted from 250 mg of cecal contents using E.Z.N.A. Stool DNA kit (OMEGA, Bio-Tek, USA). To identify the gut microbiota a primer set (515F/926R) served to amplify the 16S rRNA gene V4 region.

| LC-MS/MS metabolites analysis
A 100-mg sample of rat cecal contents was weighed and mixed thoroughly in an 800 μl acetonitrile/methanol (1:1 w/v) mixture, which was next incubated for 60 min at −20°C and thereafter centrifuged at 16,000 rpm at 4°C for 20 min. The upper phase was collected, vacuum-dried, and next added to 100 μl acetonitrile/water (1:1 w/v) for analysis in a 1290 Infinity LC system (Agilent, USA) linked to a Pegasus HT TOF mass spectrometer (LECO, USA). An ACQUITY UPLC BEH Amide 1.7 μm, 2.1 mm × 100 mm column was served to separate the single compounds. A 5.0 μl injection volume and a 25°C column temperature were used. The flow rate was 0.3 ml/min. LC-TOF/MS raw data were first analyzed by MSDIAL software using the XCMS database, including raw peak extraction, data baseline filtering and calibration, peak alignment, deconvolution analysis, peak identification, and peak area integration. Subsequently, normalized data were input into the SIMCA 14.1 software package (Umetrics AB, Umea, Sweden). After centralizing means and scaling unit variances, multivariate analyses were performed, such as orthogonal partial least squares discriminant analysis (OPLS-DA), and principal component analysis (PCA), to visualize metabolic differences between the three groups. Eventually, searching the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) allowed to characterize the differentially expressed metabolites.

| Statistical analysis
The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated via the formula: HOMA-IR = fasting insulin × fasting glucose/22.5 (Huang et al., 2017;Lin et al., 2019;Wang, Lu, et al., 2020). Each treatment group had at least three replicates (n = 3), and each experiment was repeated three times. All data values were expressed as means ± standard deviations. One-way analysis of variance was performed using SPSS version 20.0 and Origin 8.5 software. Duncan's multiple-range test served to assess any differences among groups. Values of p < .05 were taken as statistically significant. Figure 1a shows the effect of CAP + DFs on the body weight of rats fed a high-fat diet. The body weight in both the HFC and HFBM groups was significantly lower (−29.8% and −40.3%, respectively) than that in the HF group. There was no significant difference between the HFC and HFBM groups. Figure 1b shows the effects of CAP + DFs on food intake at various stages in rats fed a high-fat diet. In the first week, there were no differences in food intake among the HF, HFC, and HFBM groups.

| Effects of CAP + DFs on food intake and body weight of rats on high-fat diet
In the second week, the food intake of the HFC and HFBM groups was significant lower (−19.8% and −16.4%, respectively) than that of the HF group. In the third week, the food intake of the HFC and HFBM groups was significantly lower (−19.9% and −12.3%, respectively) than that of the HF group. Moreover, the HFBM group food intake was significantly higher (+14.5%) than that of the HFC group.
In the fourth week, there was no significant difference in food intake in the HFBM group as compared with the HFC or HF groups, albeit that of the HFC group was lower than that of the HF group. Table 1 shows the effects of CAP + DFs on the blood biochemical indices of rats fed a high-fat diet. Total cholesterol (TC) values in the F I G U R E 1 Effects of capsaicin plus dietary fibers on body weight (a) and food intake (B) of rats on a high-fat diet. A, HFC group versus HFBM group; b, HF group versus HFC group; c, HF group versus HFBM group; (one-way ANOVA followed by Dunnett's test; p < .05; n = 6). HF, high-fat diet group; HFC, high-fat diet group treated with CAP; HFBM, high-fat diet group treated with CAP plus dietary fibers HFBM and HFC groups were significantly lower (−49.8% and − 29.6%, respectively) than in the HF group. The TC value of the HFBM group was significantly higher than the HFC group's. Compared with the HF group, triglyceride (TG) levels were decreased in the HFC and HFBM groups (−34.2% and −32.9%, respectively). Low-density lipoprotein cholesterol (LDL-C) levels did not differ between the HFC and HF groups. Moreover, TG levels in the HFBM group were significantly lower than the HF group's. Compared with the HF group, high-density lipoprotein cholesterol (HDL-C) was significantly increased in the HFBM and HFC groups (+51.3% and +46.3%, respectively). With respect to the HF group, fasting plasma insulin and glucose levels, and HOMA-IR values were all decreased in the HFC and HFBM groups.

| Effect of CAP + DFs on SCFA content in the cecum of rats fed a high-fat diet
According to Table 2, the HFBM group exhibited most of the contents' changes, that is, acetic acid, +13.8% versus the HF group; nvaleric acid increased in both the HFBM and HFC groups; i-valeric acid, +25.1% versus the HF group; butyric acid increased versus the HF and HFC groups; total SCFAs, +45.8% versus the HF group. And, for the HFC group, total SCFAs increased by +29.3% versus the HF group. Finally, propionic acid levels were alike in the three groups.  Figure 2b shows the Shannon index analysis of α diversity among gut microbial samples from rats fed a high-fat diet. Thus, in the HFBM group the α diversity was increased versus that of the HF and HFC groups. Conversely, in the HFC group the α diversity was slightly decreased versus the HF group. Figure 2C shows the β diversity analysis using Bray-Curtis (BC) distance of gut microbial samples from rats fed a high-fat diet. The β diversity of the HFBM group was increased versus those of the HF and HFC groups. However, the HFC group difference was not significantly higher than that of the HF group. Results of both α and β diversity analyses indicated that the gut floras of SD rats treated with CAP significantly changed. This was especially true with the CAP + DFs treatment, which resulted in significantly increased α and β diversities.

| Effects of CAP + DFs on gut microbiota 16 s RNAs of rats fed a high-fat diet
The gut flora compositions at the level of phylum are shown in Figure 2D. The five most abundant phyla were Firmicutes, Verrucomicrobia, Bacteroidetes, Actinobacteria, and Proteobacteria.
Compared with the HF group, Firmicutes significantly decreased, while Bacteroidetes significantly increased, in the HFBM group.
Verrucomicrobia significantly increased in both the HFC and HFBM groups. Figure 2e shows the gut flora compositions at the genera level.
The abundance of Allobaculum genus was significantly more represented in the HFBM group than in both the HFC and HF groups. The abundance of Bacteroidales S24-7 genus was significantly higher in the HFC and HFBM groups than in the HF group. The Akkermansia genus abundance was higher in the HFC and HFBM groups than in the HF group-that of the HFBM group being slightly but not significantly higher than the HFC group's. The abundance of the Blautia genus was significantly higher in the HF group than in the HFC and HFBM groups.
The Desulfovibrio genus abundance was significantly higher in the HFC group than in both the HF and HFBM groups. Finally, the Desulfovibrio abundance in the HFBM group was lesser than the HF group's.

| Metabolomic analysis of cecal contents in rats fed a high-fat diet added with CAP + DFs
According to the comparison of the detected ion peaks and to the referenced database, a total of 661 compounds were detected in the cecal contents of each treatment group. Their Variable Importance for the Projection (VIP) values were calculated according to the OPLS-DA model. When VIP >1, the difference reached a significant level. A total of 39 kinds of glucose and lipid metabolites were screened. Figure 3a shows the heatmap of hierarchical cluster analysis of such cecal  Pearson correlation heatmap analysis showed that a link existed between gut microbiota genera and metabolites (Figure 3b).

| DISCUSS ION
CAP and DFs are two natural foods that regulate lipid metabolism abnormalities (Wang, Tang, et al., 2020;Zhang, Xiao, et al., 2020) In this study, CAP and DFs were added to the high-fat diet of rats. After giving this diet for a set time period, the rats' blood composition, intestinal flora, and metabolites were analyzed. In both the HFBM and HFC groups the rats' body weights were lower (p < .05) than in the HF group. This indicated that CAP helped reduce body weight. From the second week onward, the food intake by the HFC and HFBM groups was lesser (p < .05) than that by the HF group, suggesting that CAP reduced rats' appetite. Our results are consistent with those of Wang et al. (Wang, Tang, et al., 2020) who found that CAP played a role in weight loss by activating genes, such as PYY, which suppressed appetite.
Generally, obesity somewhat correlates with lipid disorders.
Four blood lipids-TC, TG, LDL-C, and HDL-C-are important indicators used to reveal changes in blood lipids (Chen et al., 2021).
Earlier studies showed that HDL-C levels negatively correlate with carotid atherosclerosis progression (Hedayatnia et al., 2020;Teis et al., 2021), while higher LDL-C levels and lower HDL-C levels might correlate with cardiovascular diseases (CVDs) such as stroke (Zhang, Wei, et al., 2020). This LDL-C/HDL-C ratio was proved to be more valuable than any single lipid component-especially LDL-C-to predict CVDs risk (Hedayatnia et al., 2020;Zhang, Wei, et al., 2020).
Here, we analyzed the blood indices of rats fed a high-fat diet.
Our results indicated that there was no significant difference in LDL-C levels in rats on a high-fed diet added with CAP alone.
Conversely, CAP + DFs significantly (p < .05) reduced blood LDL-C levels. However, in the HFC and HFBM groups HDL-C levels were significantly (p < .05) higher than in the HF group, revealing another effect of CAP alone or + DFs. Notably, a high-fat diet increases TC and TG levels in the body. In our study, CAP decreased both TC and TG levels. This was especially true with the CAP + DFs, in which TC inhibition was associated with a better prognosis of an abnormal lipid profile than in the CAP alone group.
Insulin resistance related to glucose is a metabolic defect most  In our study, acetic acid, butyric acid, and i-valeric acid were significantly increased in rats fed CAP + DFs (p < .05), indicating that this treatment may promote energy consumption by regulating the host metabolism's and immune system, and by suppressing inflammation in the gut. This was especially true for CAP + DFs, which promoted the highest production of butyric acid. These results suggested that the CAP + DFs regimen more efficiently promoted the accumulation of SCFAs. This may be due to the fact that DFs could provide a greater number of digestive substrates to gut microbes, thereby promoting the DFs' conversion into SCFAs. We recall here that the accumulation of SCFAs is one of the mechanisms decreasing TC, TG, and LDL-C levels and increasing HDL-C levels (Fei et al., 2020;Li & Pan, 2020).
The intestinal microbiota composition of the rats fed CAP + DFs significantly differed from those of the other groups. CAP + DFs increased the diversity of the gut microbial community g. The Literature reports that the Allobaculum genus is a probiotic and belongs to the Mycoplasma family (Di et al., 2019). Allobaculum produces SCFAs . Earlier studies showed that Allobaculum abundance is related to increases or decreases in body weight (Di et al., 2019). Compared with the HF group, gut abundance of Allobaculum genus was significantly (p < .05) high in rats fed either CAP + DFs or CAP alone. Moreover, Allobaculum abundance was greater with CAP + DFs than with CAP alone, indicating that the former treatment was more effective on weight loss. This view consists of the weight changes observed with the two treatments.
Although the weight difference did not reach significance, the mean body weight of rats on a high-fat diet added with CAP + DFs was slightly lower than that of rats on high-fat diet plus CAP alone. The lack of statistical significance between the HFC and HFBM groups might have been due to the too brief duration of the feeding cycle, which did not affect enough the differences in body weight.
Being among the dominant bacteroid family members, the Bacteroidales S24-7 genus is a strain of fermentation bacteria that degrades carbohydrates into SCFAs Monk et al., 2017). It usually degrades complex polysaccharides into acetic, propionic, and succinic acids (Ormerod et al., 2016). In this study, either CAP + DFs or CAP alone significantly (p < .05) increased the gut's abundance of the Bacteroidales S24-7 strain in rats fed a highfat diet. This might be one of the reasons why the accumulation of short-chain fatty acids was more abundant. The results of this study are in line with previous ones, which found that the abundance of the intestinal Bacteroidales S24-7 genus was improved by CAP gavage in rats fed a high-fat diet (Wang, Tang, et al., 2020).
The bacterial Akkermansia genus, a novel strain of probiotics, resides in the gut and exerts lipid-lowering effects. Previous studies showed that Akkermansia's abundance negatively correlates with some metabolic disorders of humans and mice such as inflammatory bowel disease, obesity, autism, and type 2 diabetes, etc. (Meng et al., 2020).
Because of this, it was important exploring CAP's effects on an intestinal microbiome member like Akkermansia (Shen et al., 2017;Wang, Tang, et al., 2020). Our results showed that adding CAP + DFs or CAP alone to a high-fat diet significantly (p < .05) increased versus the HF group the rats' gut abundance of Akkermansia. This effect was more intense in the HFBM than in the HFC group indicating that CAP + DFs may achieve better lipid-lowering effects.
Desulfovibrio is a genus of sulfate-reducing bacteria that stimulates the gut immune response promoting an inflammation that in severe cases can lead to colon cancer (Figliuolo et al., 2017;Grubb et al., 2020). The abundance of Desulfovibrio genus in the HFBM group was significantly (p < .05) lower than in the HFC group, indicating that adding CAP + DFs effectively reduced the abundance of the harmful Desulfovibrio bacteria, thereby reducing colitis and the risk of colon cancer.
Metabolomics are a group of scientific methods aimed at disclosing all the biological fingerprints associated with metabolites, molecular intermediates, and products of metabolism (Li, Qin, et al., 2019).
Metabolic profiling provides a direct, instantaneous snapshot of the physiology of the targeted cell and organism (Kalim & Rhee, 2017).
Using metabolites' analysis, we observed a series of change in responsive metabolites and screened out 18 glucose and lipid metabolites. We found that CAP + DFs increased (p < .05) the levels of arginine, Candesartan, and Irbesartan in cecum. Arginine plays a key role in the treatment of intestinal inflammation, improving gut microbiome, and reducing both oxidative stress and neutrophil infiltration (Bowerman et al., 2020;Zhang & Li, 2014 (Li & Yao, 2018). The level of candesartan and irbesartan were increased in the cecum of rats fed CAP + DFs suggest that this regimen may prevent hypertension.
Tryptophan is an essential amino acid owning an indole ring that is present in dietary proteins (Kałużna-Czaplińska et al., 2019).
Although tryptophan is the least abundant amino acid in proteins and hence in cells, it is a precursor of the biosynthesis of a large number of microbial metabolites. In vitro studies showed that some patients produced, transported, and catabolized lesser amounts of tryptophan. This indicated that tryptophan plays a crucial role in human T cell differentiation, immune regulation, and neurological function (Sandgren & Brummer, 2018). Tryptophan's levels in the cecal contents of rats fed CAP alone or CAP + DFs were significantly (p < .05) increased, suggesting that either treatment may improve overall immune function.
PC, LPE, and LPC are involved in glycerophospholipid metabolism . In our research, Lpe:16:0, Lpe18:1, Lpe18:2, Lpe18:3, and Lpc18:3 were all significantly (p < .05) decreased in rats fed CAP alone or CAP + DFs. Interestingly, when compared with CAP alone, both Lpe18:1 and Lpc18:3 levels were significantly (p < .05) higher in rats fed CAP + DFs. This result has been due to the increased variety of intestinal flora, which promotes the maintenance of intestinal phospholipid levels by some nonpathogenic bacteria (Zou et al., 2020).

| CON CLUS IONS
There are large numbers of complex microbial communities in the human gut. The interaction between gut microbes and the host forms a network system that maintains bodily health. We studied the effects of adding CAP + DFs on the intestinal microbiome and metabolism on rats kept on a high-fat diet. Our results showed that CAP + DFs added to a high-fat diet (1) regulated rats' lipid metabolism better than CAP alone did; (2) improved the diversity of intestinal microbiome, optimizing its composition, while reducing the abundance of Desulfovibrio genus bacteria in rats fed a high-fat diet; and (3)  Education Commission (KJ1726396).

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict of interest. They also declare that have no financial and personal relationships with other people or organizations that can inappropriately influence their work; that there is no professional or other personal interest of any nature or kind in any product, service, and/or company that could be construed as influencing the data presented in, or the review of, the manuscript entitled, "Capsaicin combined with dietary fiber prevents high-fat diet-associated aberrant lipid metabolism by improving the structure of intestinal flora."

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.

E TH I C S S TATEM ENT
All procedures followed were in accordance with the ethical stand-